Effect And Regulating Mechanism Of Stigmasterol On Neuroinflammation In Alzheimer’s Disease

Currently in China,there are 15.07 million dementia patients among people aged 60and over,of which 9.83 million are living with Alzheimer’s disease（AD）,accounting for65%.With the in-depth studies of the pathologies in AD,neuroinflammation has become one of the potential therapeutic targets.Phytosterols are a group of natural products with a variety of physiological activities,and have anti-inflammatory effects in various disease models.Stigmasterol is one of the common dietary phytosterols.Studies have shown that it may have positive effects on neuroinflammation in AD as well as gut microbiota dysbiosis.However,there is no current study concerning the direct regulatory mechanisms of stigmasterol on neuroinflammation or the indirect modulation through gut microbiota.Therefore,this article took stigmasterol as research object,combined with APP_swe/PS1_{d E9}mice model,in vitro blood-brain barrier model,and AβO-induced microglial inflammatory response model,using GC-MS,proteomics and 16S r RNA analysis to investigate the effects of stigmasterol on AD and neuroinflammation,the efficiency of stigmasterol traversing blood-brain barrier and its effects on gut microbiota,and the potential mechanisms of stigmasterol regulating inflammatory response in microglia.The main research contents and results are summarized as follows:（1）The effects of stigmasterol intake on cognition,pathological features and neuroinflammation in AD were investigated using APP_swe/PS1_{d E9} mice.Gavage for 4 weeks of 50 mg/kg stigmasterol improved spatial learning and memory of APP/PS1 mice,while reducing the content of Aβ₄₂ in the brain.The composition and function changes of brain proteins were further studied by quantitative proteomic.Gene Ontology revealed that stigmasterol affected the learning and memory,abnormal protein formation and neuroinflammation in APP/PS mice.Further analysis of the differential expressed proteins showed that microglia mediated inflammatory response-related proteins were changed significantly.Studies in inflammation showed that stigmasterol intake effectively reduced the secretion of inflammatory factors TNFαand IL-1βin APP/PS1 mice,and inhibited the excessive activation of microglia and astrocytes in the cortex and hippocampus.Stigmasterol also had regulatory effects on NF-κB signaling and NLRP3 inflammasome.（2）The direct and indirect effects of stigmasterol on neuroinflammation were studied in terms of blood-brain barrier permeability and gut microbiota.The intestinal absorption rate and brain/blood ratio of stigmasterol in C57BL/6J mice were 1.5%and 4.5%,respectively.In the co-culturing blood-brain barrier model,absorption rate of 50μM stigmasterol was 3.12%,and the P_app was 9.67×10^-7 cm/s,indicating a difficult-to-absorb pattern.Meanwhile,with the increase of stigmasterol concentration,the efflux rate decreased.After continuous gavage of 50 mg/kg stigmasterol for 4 weeks,the stigmasterol content in the brain of APP/PS1 mice increased by 0.98 times.At the same time,stigmasterol intake ameliorated gut microbiota diversity,dysbiosis and intestinal inflammation in APP/PS1 mice,while reducing the relative abundance of pro-inflammatory Alloprevotella,Parasutterella and Desulfovibrio fairfieldensis,and increased the anti-inflammatory Lactobacillus reuteri.The relative abundance of four differential bacteria was significantly correlated with systemic inflammation,neuroinflammation,pathological features and cognitive function in APP/PS1 mice.（3）Using AβO-induced BV2 cells as a model of microglial inflammatory response,the regulation and underlying mechanism of stigmasterol on microglial inflammation were studied.The viability and inflammatory cytokines secretion of BV2 cells after different concentrations of AβO treatment were measured,and the modeling concentration of AβO was determined to be 1μM.In this model,10μM and 20μM stigmasterol significantly reduced the secretion of pro-inflammatory cytokines TNFαand IL-1βinduced by AβO,while increasing the secretion of anti-inflammatory cytokine IL-10,in addition,stigmasterol was able to reduce expression of CD86 receptor on the cell surface,thereby inhibiting AβO-induced polarization to M1 type.Studies on inflammatory pathways found that 20μM stigmasterol up-regulated the phosphorylation of AMPK,thereby inhibiting the activation of NF-κB pathway and NLRP3 inflammasome caused by AβO,thus reducing the secretion of pro-inflammatory factors TNFαand IL-1β.